Transient changes in effluent pH can occur during ion-exchange chromatography. These changes are sometimes referred to as “pH excursions” or “pH transitions” and can be unexpected and uncontrolled. Under some conditions, considerable pH excursions can occur in ion-exchange systems even if the pH of the solution entering the chromatography column is the same as the pH of the column equilibration solution. This phenomenon is described, for example, in Ghose et al., Biotechnol. Prog. 18:530-537 (2002). Pabst and Carta, J. Chromatography A 1142:19-31 (2007) also describe this phenomenon, noting that uncontrolled pH transitions occur in cation exchange columns during equilibration and salt elution when the stationary phase contains weak acid groups even if the mobile phase is buffered and the buffering species does not interact with the stationary phase.
It has previously been essentially impossible to generate a collinear pH/conductivity gradient in cation exchange chromatography in part due to pH excursions. On cation exchangers, the pH drops as soon as sodium chloride is introduced. Depending on the choice and concentration of buffering species, the charge characteristics of the exchanger, and the concentration of sodium chloride introduced, many column volumes of buffer can be required before pH normalizes. The pH increase will not parallel conductivity, since conductivity rises linearly from the start of the gradient. This causes aberrant elution profiles that can substantially reduce the quality of the fractionation. The corresponding phenomenon is also observed in anion exchange chromatography in which the pH increases initially in an aberrant non-linear fashion in an increasing salt gradient.
The methods described herein address these and other issues.